Solid-phase capture and affinity purification have a wide variety of applications in research, clinical, and pharmaceutical laboratories. For the batch analysis of multiple samples, a capture reagent (e.g., an antibody) can be immobilized on the bottom of microtiter plate wells to bind a molecule of interest (e.g., the corresponding antigen). This system provides a relatively small surface area for capture and a small working volume during binding and washing of the target molecule. Such assays allow for the determination of the presence of a molecule in a sample, but do not typically allow for the isolation of a sufficient quantity of a molecule to perform further tests. Moreover, it is difficult to remove the molecule of interest from microtiter plates. Microtiter plates are typically made of clear, polystyrene plastic to allow for colormetric assays to be performed. Polystyrene is not stable in the presence of a variety of organic compounds frequently used for elution. Additionally, the plates cannot be heated to elute the samples into loading buffer for analysis of the samples by SDS-polyacrylamide gel electrophoresis (PAGE).
A higher surface area for solid phase capture can be obtained by the use of microbeads coated with the capture reagent. Microbeads made of any of a number of materials for attachment of a variety of capture reagents have been reported. One of the most commonly used polymer microbeads are agarose microbeads. Frequently, either protein A or protein G is attached to the beads through a linking reagent such as cyanogen bromide for capture of antibodies on the beads. The antibodies captured on the protein A or protein G coated beads can subsequently be used as capture reagents for capture of the antigen corresponding to the antibody.
A number of other microbeads have been reported in the literature including magnetic beads (i.e. polystyrene-coated paramagnetic iron beads, M-450 Dynabeads) for the capture of monoclonal antibodies (Quitadamo and Schelling, Hybridoma, 17:199-207. 1998; incorporated herein by reference); particulate nitrocellulose for the capture of a variety of proteins (Hammerl et al., J. Immunol. Meth., 165:59-66. 1993, incorporated herein by reference); protein A Superose (crosslinked agarose) beads (Leibl and Erber, J. Chromatogr., 639:51-56.1993, incorporated herein by reference), protein A polyhydroxyethylmethacrylate beads (Denizli et al., J. Chromoatogr. B, 668:13-19.1995, incorporated herein by reference), and alginate-chitosan beads (Albarghouthi et al., Int. J. Pharm., 206:23-34. 2000, incorporated herein by reference) for the capture of immunoglobulins; and glass beads for the capture of antibodies (Phillips, J. Biochem. Biophys. Methods, 49:253-262. 2001, incorporated herein by reference) and single stranded DNA (Walsh et al., J. Biochem. Biophys. Methods, 47:221-231, incorporated herein by reference). A number of additional beads and linking reagents are shown in Table 1 and Table 2 from Nisnevitch and Firer (J. Biochem. Biophys. Methods. 49:467-480. 2001, incorporated herein by reference). The higher surface area of the beads as compared to the microtiter plate allows for the capture of a substantially larger quantity of the molecule of interest. Such affinity purification methods are powerful techniques and can be used for a number of purposes including determining the presence and quantity of a macromolecule in a sample, determining the rate of a macromolecule's synthesis and degradation, identifying complexes of macromolecules and purifying small amounts of macromolecules.
The use of these techniques is limited both by the cost of the reagents required (e.g. protein A- or protein G-agarose) and the amount of effort required to perform the assays. As opposed to microtiter plates that can be filled with wash buffer using a multichannel pipettor and the buffer removed by inversion of the plate, samples purified on beads must be handled individually and with care. Each individual tube must be filled with buffer, mixed to resuspend the beads, centrifuged briefly to collect the beads at the bottom of the tube and the buffer aspirated carefully so as not to aspirate the beads. This limits the number of samples that can be processed by an individual at one time and makes automation of the process difficult. Due to the number of washes required, it is not practical to process more samples than can be placed in a single microfuge rotor, typically 18. Moreover, many macromolecules and macromolecular complexes are labile. To increase the stability of the molecules and complexes, immunoprecipitation assays are often performed in a cold room (4° C.), which is both uncomfortable and impractical.
Solid phase capture assays using beads may also be performed by column chromatography. The macromolecule of interest is captured on beads coated with the capture reagent by batch binding or by passing a mixture containing the molecule of interest over a column. Although such methods are somewhat more amenable to automation, elution of the molecule of interest from the column requires a relatively large amount of buffer, frequently resulting in a solution of the molecule of interest that must be concentrated before further analysis. This is time consuming and can result in deterioration of the sample.